To validate the loss of interaction between FAM161A and variant POC1B in mammalian cells, we cotransfected hTERT-RPE1 cells with constructs encoding wild-type and variant mRFP-POC1B together with PalmMyr-CFP-FAM161A (Figures 5B–5D; Figures S7A–S7C). The PalmMyr tag provides residues for palmitoylation and myristoylation, which both induce membrane association of the protein of interest.29 This can subsequently be visualized by fluorescence microscopy of the expression of the fluorescent CFP tag. Coexpression of FAM161A and POC1B showed complete colocalization of the encoded proteins at the plasma membrane, basal body, and association with the microtubule network. When either one of the mutations was present in the POC1B construct, the colocalization with FAM161A was lost completely. PalmMyr-CFP-FAM161A then maintained its membrane, basal body, and microtubule association, but the localization of variant POC1B was cytosolic without enrichment at specific subcellular sites (Figures 5C and 5D; Figures S7B and S7C).